Field of the Invention
This invention concerns a novel human AZU-1 gene, mutants, variants and fragments thereof, and protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. In particular, this invention concerns identifying, isolation and characterization of novel AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. Additionally, the invention concerns findings that AZU-1 and AZU-2 genes exhibit tissue-specific expression profiles and that AZU-1 gene expression in tumor cells is low or absent. The invention further concerns recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes. The invention also concerns monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.
The evolvement of breast cancer is a multistep and cumulative process and understanding of the genetic and phenotypic alterations in successive steps is essential for designing therapeutic interventions and diagnostic assays.
In the human body, the epithelial component of the breast is embedded in the stroma and forms a branching ductal structure that emanates from the nipple, repeatedly bifurcates, and terminates in lobules, alveoli, and end buds. Although stroma accounts for  greater than 80% of the breast volume, approximately 95% of the cancers produced in the breast are of epithelial origin. To elucidate the advancement of human breast cancer, a functionally relevant cell culture model is required. The differences in breast tissue compartmentalization, phenotypic characteristics, and the mutagenic frequency between human and rodents underline the need to develop a human breast cell model.
An unconventional spontaneously-transformed HMT-3522 cell lines was described recently in Cancer Research, 56:2039 (1996) where the immortalized human mammary epithelial cells (S1) established from fibrocystic breast tissue was propagated in chemically defined medium described in In Vitro Call. Dev. Biol., 23:181 (1987). S1 cells were near-diploid and expressed luminal epithelial cell differentiation markers cytokeratin-18 and sialomucin. Genetic changes such as p53 point mutation (Exp. Cell. Res., 215:380 (1994)) and c-myc amplification (Cancer Research, 52:1210 (1992)) have already been noted in later passages, i.e.,  greater than 50 of the nonmalignant S1 cells. In passage 118, cells were adapted to grow in epidermal growth factor (EGF)-free medium and a new EGF-independent subline (S2, premalignant) was isolated (ibid).
Alterations in gene expression of EGF receptor, transforming growth factor (TGF)-xcex1 and c-erb-B2 were seen in S2 cells. The established S2 cell line underwent cytogenetic evolution and exhibited genomic instability and heterogeneity (Cancer Genetics and Cytogenetics, 78:189 (1994)). Nevertheless, S2 cells remained nontumorigenic until passage 238. At that time, it was able to induce tumor growth in nude mice. Following second round of mouse transplantation, another subline (T4-2, tumorigenic) existed as a relatively homogenous malignantly-transformed cell population. One extra copy of chromosome 7p which harbors EGF receptor gene was found in the T4-2 cells (Cancer Res., 56:2039 (1996)).
Detection and suppression of cancerous tissue growth is of extreme interest and importance. It is, therefore, a primary objective of this invention to provide means for identification, detection and suppression of cancerous growth in breast tissue.
All patents, patent applications and publications cited herein are hereby incorporated by reference.
One aspect of the current invention is a human AZU-1 gene or a variant, mutant, or fragment thereof.
Another aspect of the current invention is a nucleotide sequence of AZU-1 and AZU-2 genes, identified as SEQ ID NO: 1 and SEQ ID NO: 2.
Another aspect of the current invention is a DNA sequence identified as SEQ ID NO: 1 encoding a protein comprising the amino acid sequence identified as SEQ ID NO: 3.
Still another aspect of the current invention is a protein of the amino acid sequence identified as SEQ ID NO: 3.
Still yet another aspect of the current invention is a protein encoded by AZU-1 gene, or by a variant, mutant or fragment thereof, or any protein containing said protein encoded by AZU-1 gene, variant, mutant, or fragment thereof, or any protein which shares homology with AZU-1 encoded protein or AZU-2 encoded protein, variant, mutant or fragment thereof.
Still another aspect of the current invention is a protein encoded by the AZU-1 gene, variant, mutant, analog or fragment thereof acting as a tumor suppressor or a marker of malignancy progression and tumorigenic reversion.
Still yet another aspect of the current invention is a method for diagnosis and detection of progression of human breast cancer by detecting presence and quantity of a protein identified as SEQ ID NO: 3 or a homolog thereof in human breast cells or tissue or by detecting expression of AZU-1 gene, mutant, variant, fragment thereof by in situ hybridization or RT-PCR.
Still yet another aspect of the current invention is a diagnostic method for detection of the presence of AZU-1 protein in human breast cancer by treating a biopsy sample of a subject patient with a polyclonal or monoclonal AZU-1 antibodies or detection of the level of AZU-1 message.
Yet another aspect of the current invention is a method for prevention or treatment of human breast cancer by providing a subject in need thereof a therapeutically effective amount of a protein identified as SEQ ID NO: 3 or homolog thereof, able to act as a tumor suppressor of human breast cancer cells.
Still yet another aspect of the current invention is a tissue targeted gene therapy for treatment of human breast tumor.
Still another aspect of the current invention is an ELISA kit for detection of expression of AZU-1 gene or a variant thereof by detecting presence or absence of AZU-1 encoded protein with AZU-1 monoclonal or polyclonal antibodies.
Still another aspect of the current invention is a message detection kit for detection of expression of AZU-1 gene or a variant thereof by bDNA technology (Quantigene Gene Expression Assay, Chiron Corp., Emeryville, Calif., in situ hybridization or RT-PCR by detecting presence or absence of AZU-1 message.